Melanoma is a common, aggressive malignancy characterized by genetic heterogeneity and resistance to therapy. To improve patient survival, new molecular targets and treatment strategies are needed. The long- term goal of this proposal is to test the hypothesis that inhibiting the cancer-critical survival protein HSP70 constitutes an effective new strategy for melanoma therapy. The stress-inducible HSP70 molecular chaperone is an attractive target for several reasons: this protein is markedly overexpressed in melanomas relative to normal melanocytes, and considerable evidence points to the importance of the mulfi-functional HSP70 chaperone in promoting melanoma tumorigenesis. HSP70 plays a central role in maintaining protein quality control and promoting tumor cell survival in response to a variety of endogenous and exogenous stresses. We have identified and characterized a novel small molecule (2-phenylethynesulfonamide, or PES) that selectively binds to HSP70 and inhibits its function. Our studies demonstrate that PES is preferentially cytotoxic to melanoma tumor cells relative to non-transformed cells or primary melanocytes. In the proposed study, we will use biochemical and structural biology analyses to probe the mechanism of action of PES. We will perform a structure-activity relationship analysis for this compound, and pursue derivatives with enhanced cytotoxicity and HSP70-inhibition. We will elucidate genotype(s) of melanoma with particular sensitivity to PES, and assess potential synergy with other, melanoma relevant compounds. We will better elucidate the pathways of cytotoxicity, and define pathways of resistance. And finally, we will test the impact on HSP70 inhibition on melanoma initiation and progression, in transgenic and patient- derived xenograft models. Taken together, the integrated research plan takes advantage of the unique structural biology, chemistry, and mouse models expertise of the program Investigators in order to further develop an exciting new strategy for melanoma therapy. As such, the proposed investigations will begin to address a major unmet clinical need. RELEVANCE (See Instructions): Melanoma is a major public health problem for which there are few effective treatment options. Because the HSP70 molecular chaperone promotes melanoma tumorigenesis through multiple signaling and protein quality control networks, it is recognized as a promising new target for therapy. The proposed research plan takes advantage of our recent identification of a novel class of small molecule HSP70 inhibitors to test a promising strategy for treating melanomas based on inhibiting this cancer-critical survival protein.